1.Role of Membrane Interactions on the Mechanism of alpha-Synuclein Amyloid Formation Understanding the environmental factors affecting the aggregation of alpha-synuclein (alpha-syn) is of great importance because the accumulation and deposit of alpha-syn are intimately connected to Parkinsons disease etiology. Membrane interactions are of particular interest because alpha-syn localizes near synaptic vesicles and mitochondrial membranes in vivo. Specifically, the protein undergoes disordered-to-helical structural changes with the addition of membrane mimics such as SDS micelles and upon binding to anionic phospholipid vesicles of varying size and composition. To develop a detailed understanding of how membranes influence alpha-syn conformation, site-specific probes of protein conformational heterogeneity and polypeptide-membrane interactions are necessary. Fluorescence spectroscopy is particularly suited for this application because of the availability of environmentally sensitive fluorophores and the ease of performing experiments near physiological temperatures and concentrations even down to a single molecule. In this study, we have employed anionic SUVs and SDS micelles as membrane mimics to investigate membrane-induced conformational changes by fluorescence as well as circular dichroism (CD) spectroscopy. Tryptophan was substituted at four different aromatic residues (F4W, Y39W, F94W, and Y125W) to report information on local polypeptide environment and conformational heterogeneity between SUV- and SDS-micelles-bound alpha-syn. Furthermore, insights into the role of surface coverage were extracted from saturable equilibrium binding curves for all alpha-synucleins in the presence of SUVs. Measurements of steady-state and time-resolved fluorescence of single tryptophan-containing alpha-syn variants have revealed distinct phospholipid vesicle and micelle interactions at residues 4, 39, 94, and 125. Our CD data confirm that Trp mutations do not affect alpha-syn membrane binding properties saturating at an estimated lipid-to-protein molar ratio of 380 or approximately 120 proteins covering 7% of the surface area of an 80 nm diameter vesicle. Fluorophores at positions 4 and 94 are the most sensitive to the lipid bilayer with pronounced spectral blue-shifts (W4: 23 nm;W94: 10 nm) and quantum yield increases (W4, W94: 3 fold) while W39 and W125 remain primarily water-exposed. Furthermore, measurements of fluorescence decay kinetics reveal the presence of protein conformational heterogeneity in the bilayer, suggesting that the both W4 and W94 exhibit high membrane affinity. Notably, both of these sites have not been characterized previously in the vesicle-bound alpha-syn structure. With this approach, we can determine the crucial protein-to-membrane conditions and key sites of interaction that promote protein aggregation and ultimately, monitor membrane-mediated amyloid formation processes. 2. Copper(II) Binding to alpha-Synuclein Although recent work points to a genetic component to PD involving the accumulation and deposit of a neuronal protein, alpha-syn, the sporadic form of the disease is far more common and possibly connected to environmental factors that promote oxidative stress and aberrant redox-active metal metabolism. For example, selective accumulation of fibrils in dopaminergic neurons in PD has been attributed to the presence of easily oxidizable catechols that stimulate protein cross-links, as well as to increased iron concentration in Lewy bodies and copper in cerebrospinal fluid of PD patients. Furthermore, metal-enhanced oxidative oligomerization has been observed for alpha-syn in vitro, and, specific metal-protein interactions have been proposed to be critical in other neurodegenerative diseases involving amyloidogenic biomolecules such as amyloid beta-peptide (Alzheimers disease), prion protein (spongiform encephalopathies), and superoxide dismutase (amyotrophic lateral sclerosis). A difficult issue to resolve is whether metal ions perturb protein structures and thereby alter functions, or whether metal-protein complexes directly participate in the production of reactive oxygen species, or whether both mechanisms are at work. In our prior efforts, we have exploited a fluorescent amino acid, Trp, as a site-specific probe of Cu(II)-protein interaction. In particular, we found that Trp4 is the most responsive reporter of Cu(II), indicating a high-affinity N-terminal site with an apparent dissociation constant (100 nM, pH 7). (JACS 2008) We also demonstrated that the substitution of His50 with a Ser residue has little effect on the Cu(II)-affinity of the protein. Currently, we have identified the minimal amino acid sequence necessary to preserve Cu(II) affinity by employing a series of synthetic Trp-containing alpha-syn peptides: alpha-syn1-10, alpha-syn1-6, and alpha-syn1-4. Surprisingly, the removal of more than 130 residues has relatively little effect on metal-protein interactions;the titration curves for the three peptides and proteins are nearly indistinguishable. Indeed, our data strongly indicate that only the first four residues are essential to preserve Cu(II) affinity. These results clearly support our initial proposal that His50 is not required for N-terminal Cu(II) binding. Furthermore, our data indicate that even if His50 does interact with the copper center, it would contribute minimally to the stability of the metal-protein complex because the copper is chelated by the N-terminus and backbone amides. We further evaluated the roles of specific amine ligands: Lys at positions 6 and 10 as well as the alpha-amino terminus. Consistent with our hypothesis that the essential ligands for Cu(II) reside in the first four residues, binding curves for mutant peptides containing Arg substitutions are nearly identical to alpha-syn1-10, ruling out the binding of Lys6 and/or Lys10. To assess the participation of the alpha-amino moiety, the N-terminus was acetylated on all peptides. This modification completely disrupts Cu(II) coordination and assigns the alpha-amino group as the critical ligand. Our results provide direct evidence that for alpha-syn, a free N-terminal NH2 is required for Cu(II) binding. The finding that the alpha-amino terminus is crucial for Cu(II) binding in alpha-syn leads us to suggest a known motif in Cu(II)-polypeptide interactions: Cu(II) is anchored by the free amino-terminal nitrogen and chelates to adjacent backbone amides. Our data indicate that through post-translational modification of the N-terminus, copper-protein chemistry could be modulated in vivo. Recent studies have provided evidence for considerable alpha-amino terminal acetylation (to what extent remains unclear) in alpha-syn extracted from different cellular sources. It is therefore difficult to assess the significance of N-terminal-Cu(II)-binding in alpha-syn function and its associated disease state. Our data clearly demonstrate that Trp fluorescence is a sensitive molecular probe of Cu(II) and N-terminal alpha-syn interactions. Using site-directed mutagenesis and synthetic peptides, we show that His50 has little or no contribution to the formation of the high affinity Cu(II) coordination complex. Specifically, we identify the first four residues, MDV(F/W), as the essential binding site, anchored by the alpha-amino terminus. This peptide binds Cu(II) with a submicromolar dissociation constant comparable to the full length protein. Future studies are aimed at elucidating the nature of Cu-alpha-synuclein interactions in the presence of other biomolecules, e.g. phospholipid bilayers, is necessary in defining the role of metal ions in PD, as well as other synucleinopathies.